In Vitro and In Vivo Evaluation of Poly (3-hydroxybutyrate)/Carbon Nanotubes Electrospun Scaffolds for Periodontal Ligament Tissue Engineering

Statement of the Problem: Tissue engineering was an idea, today it has become a potential therapy for several tissues in dentistry, such as periodontal disease and oral mucosa. Purpose: In this experimental study, periodontal regeneration is one of the earliest clinical disciplines that has achieved therapeutic application in tissue engineering. The aim of the present study was to prepare electrospun Poly (3-hydroxybutyrate) (PHB)/1% Carbon nanotubes (CNTs) scaffolds for periodontal regeneration. Materials and Method: 1% w/v of CNTs was added to the polymer solutions and electrospinned. Physical properties of the scaffolds were evaluated by scanning electron microscopy (SEM) and universal testing machine. Chemical characterization of the scaffolds was also assessed by Fourier-transform infrared spectroscopy (FTIR). Biological properties of the scaffolds were also evaluated in vitro by culturing periodontal ligament stem cells (PDLSCs) on the scaffolds for 10 days and in vivo by Implanting the scaffolds in rat model for 5 weeks. Results: Results showed that the scaffolds mimicked fibrous connective tissue of the (PDL). CNTs improved the mechanical properties, similar to 23-55 years old human PDL. In vitro biocompatibility study showed more attachment and proliferation of the PDLSCs for PHB/1%CNTs scaffolds compared to the PHB controls. In vivo study showed that CNTs in the scaffolds caused mild foreign body type giant cell reaction, moderate vascularization, and mild inflammation. Conclusion: The results showed that the PHB/1%CNTs composite scaffolds might be potentially useful in periodontal regeneration.


Introduction
The periodontal ligament (PDL) is a soft tissue embedded between the cementum (a thin layer of mineralized tissue covering the roots of the teeth) and the inner wall of the alveolar bone socket, to sustain and help constrain the teeth within the jaw [1]. The PDL is composed of fibrous connective tissue and a physically small, but functionally important tissue in tooth support, proprioception and regulation of alveolar bone volume [2].
Periodontal diseases are the most common inflammatory diseases, caused by plaque biofilm in the oral cavity.
For more than three decades, periodontal research has attempted to discover clinical treatment regimens that can regenerate periodontal tissues with good predictabil-ity [3]. The strategy aimed at regenerating a 3D-arrayed structure of lost PDL tissue with the connective tissue attachment. To achieve complete regeneration, the introduction of modern tissue engineering technology is anticipated in this field [3]. Tissue engineering combines the principles and methods of the life science with those of engineering to elucidate fundamental understanding of structure-function relationships in the normal and diseased tissues, to develop materials and methods to repair damaged or diseased tissue, and to create entire tissue replacements [4].
In this context, three-dimensional porous scaffolds can provide the condition, necessary for the cells to proliferate, migrate, and maintain their differentiated function by creating an empty space and function as a framework for thriving tissue [5][6]. Myriad natural and synthetic materials have been evaluated for tissue engineering scaffolds [6]. The sizes of extracellular matrix components, including porosities and the fibers diameters are at a range of nano-scales, using nano-fibers has increased in tissue engineering [7][8]. Amongst numerous methods, electrospinning process is considered a cheap method to prepare nanofibers from both natural and synthetic materials (fiber diameters from micrometer to nanometer range) [9][10]. Electrospun scaffolds offer an appropriate morphology and porosity that resembles the natural extracellular matrix (ECM) by providing a good condition for cell attachment, proliferation, and differentiation [11][12].
In the past decades, PHB is considered as a biodegradable and biocompatible material that has been applied for biological applications in medicine [13].
The challenging between combination of biomedical and biodegradable properties of PHB is a perspective tool in the design of novel medical devices and tissue engineering [14]. Although this polymer has a myriad of medicinal application and the properties necessary for use as a scaffold, it does not have enough strength, which is necessary for the three-dimensional scaffolds for hard tissue engineering [15]. Selection of a suitable material to improve the mechanical and biological properties of PHB would be imperative. Regarding this issue, carbon nanotubes have been extensively used for biological and biomedical applications in the past few years due to their unique intrinsic physical, chemical, and mechanical properties [16]. Evaluations of many researchers have shown that functionalized CNTs are able to enter the cells without toxicity, shuttling various biological molecular cargoes into the cells [17][18].
Despite these exciting findings, researchers reported the negative sides of CNTs, including those nonfunctionalized nanotubes are toxic to the cells and animals [17]. CNTs functionalization is thus, required and involves the addition of functional groups such as carboxyl or alcohol groups to the walls and ends of the nanotubes [18]. This should prevent CNTs aggregation and allow for their incorporation into polymer scaffolds [19]. Studies showed that nanoparticles of bioceramics could improve the properties of the polymers both mechanically and biologically. For significant increase in the properties of the polymers, by nanoparticles, high amounts of nanoparticles (Close to 10-20% by weight) have to be added to the basic polymer [20][21]. However, numerous studies showed that CNTs with low percentages (Close to 1-2% by weight) could significantly increase the properties of different polymers [22][23]. Jeong et al. [22] evaluated different amounts of CNTs (1%, 2.5%, 5%, and 7.5%) on polyvinyl alcohol scaffolds and revealed that 1% of the CNTs had the most improvement in mechanical properties of the scaffolds. In another study, the results indicated that the presence of only 0.5% of functionalized CNTs in poly-lactic glycolic acid scaffolds increased the tensile strength by 54% and increased the in vitro cell compatibility compared with the polylactic glycolic acid control [24].
Regarding the biological and mechanical properties of functionalized CNTs and using them as confirmatory material in the scaffolds, the aim of the present study was to prepare PHB/1%CNTs nanocomposite scaffolds via electrospinning and evaluate their structural, mechanical properties (according to the PDL), in vitro cell compatibility using human PDLSCs and in vivo tissue biocompatibility, (according to the tissue engineering parameters) for oral tissue engineering applications.

Surface wettability
The contact angle was measured with a drop on the specimen after 1 minute of drop deposition [27]. The measurements of static contact angles were mainly performed with the specimen placed on a horizontal plane; a drop of water was dropped on the surface of the sample via a precise dropper and the picture of drop on the specimen was seen using a telescope with a calibrated micrometer lens. On each occasion, at least 10 measurements were taken, and the static contact angles were determined using the Image J software.

Bioactivity evaluation
According to Kokubo and Takadama's definition of bioactivity, a bioactive material is one on which bone-like hydroxyapatite will form selectively after it is immersed in a serum-like solution [28]. In this study, SBF solution environment was used to characterize the bioactivity of the scaffolds by immersing them into the solution for four weeks. During this period, from the first to the fourth week, 5 samples were used for each group and atomic absorption spectroscopy (AAS) method (AAS-Perkin Elmer Co-A-Analyst-300) was weekly employed to measure the absorption level of Ca2+ in the SBF solution.
SEM and energy-dispersive X-ray (EDX) were applied to observe and prove the existence of sedimentary hydroxyapatite crystals on the surface of the fibers. After four weeks, X-ray diffraction (XRD) was also used to study the structure and prove the presence of hydroxyapatite on the surface of the fibers for more assurance.

PDL cell isolation
In vitro cell culture was performed using PDLSCs de-

Evaluation of PDL Cell adhesion and proliferation
Morphological study of PDLSCs grown on electrospun PHB and PHB/1%CNTs was performed by SEM after 1 and 10 days of cell culture. The cell-seeded scaffolds were collected after 1 and 10 days and entirely washed with PBS solution. Subsequently, the cells on the scaffolds were fixed by 2.5% glutaraldehyde in PBS for 1h at 4 ᵒ C. Afterwards, the specimens were dehydrated using 50-100% ethanol with 30 min each grade, dried, gold splattered in vacuum, and examined using SEM.
MTT assay was used to assess the PDLSCs' metabolic activity in the presence of intended scaffolds. Briefly, MTT solution was ready in PBS, sterilized, added to each well, and incubated for 3h at 37 ᵒ C. Then, to dissolve the red colored formazan crystals formed, we added dimethyl sulfoxide and the absorbance was measured at 570nm with ELISA plate reader.

Animal study
Ethical considerations were confirmed by the Animal Care and Use Committee of the Shiraz University of Medical Sciences (Approval No: IR.SUM.REC.1396-S1037). Sprague dawley male rats (aged 8-10 weeks; weighted 220±20g; n = 5 rats for each group) were prepared from Laboratory Animals Center of Shiraz University of Medical Sciences, Shiraz, Iran. All the rats were kept at standard room (temperature (22±2°C); humidity 55±5%; ventilation 12 times per hour and 12 hours light/dark cycle). They were fed a standard pellet diet ad libitum.

Scaffold implantation
The rats were anesthetized using 90mg/kg Ketamine 10% (Alfasan, woerden-Holand) and 8 mg/kg Xylazine 2% (Alfasan, woerden-Holand) with the eyes protecting the application of ophthalmic liquid gel (Alco Canada In., ON, Canada). The rat's back hair was shaved and sterilized using Povidone lodine 10% (Pejhan Chemic Yazd Co. Iran), 5mm incisions were cut on the dorsal section of each rat with aseptic surgery method, and cell-free scaffolds were implanted under subcutaneous pouch. The incisions were then sutured and oxytetracycline aerosol spray was topically applied to the surgery sites to prevent infection. Additionally, 0.03 mg/kg buprenorphine was administrated as a pain reliever. All the rats were carefully monitored during the study.

Histopathological analysis
The aim of histological assessment was to evaluate the biocompatibility of the scaffolds including extracellular matrix deposition, vascularization, formation of foreign body giant cell, and inflammation. At the end of the 5th week after the surgery, the rats were euthanized using CO2 inhalation in a specific chamber; the dorsal skin was carefully resected and fixed in 10% formalin. Serial 5μm thick sections were cut, and stained with hematoxylin-eosin (H&E). For the evaluation of cell infiltration, extracellular matrix deposition and vascularization (angiogenesis), micrographs were captured using Olympus microscope (model BX53F, Japan) equipped with 40x and 400x objective and evaluated by a blind pathologist.

Statistical analysis
The collected data were analyzed by one-way analysis of variance (ANOVA) and reported as mean standard deviation. All observations were confirmed by at least three independent experiments. The Data were analyzed using IBM SPSS software and the level of significance was set at p< 0.05. Figure 1 illustrates the morphology of pure and composite scaffolds. They mimicked the human PDL tissue (provided by Beertsen et al.) [29]. Presented SEM micrograph of electrospun fibers showed that the created fibers were structurally similar to the collagen fibrous structure in PDL. The obtained fibers of the scaffolds were smooth without any bead; the majority of the fibers' diameter for pure PHB was confined to the range of 180-320 nm, with a mean of 240nm ( Figure 2). This range of fiber diameter for composite scaffold increased to 400-590nm by addition of only 1% w/v CNTs. By using MALAB software program, the obtained results of porosity showed that the porosity of electrospun nano fibrous scaffolds was over 80%, which is suitable for the purposes of tissue engineering.

Mechanical characterization
The tensile strengths of pure PHB and PHB/1%CNTs are shown in Figure 4.  The results showed that the tensile strength of nanocomposite scaffold was close to tensile strength of the human PDL compared to pure PHB [30]. The tensile strength of the composite scaffold compared to pure scaffold clearly and significantly increased in the presence of only 1% CNTs.

Water contact angel
The macro-photograph of the water drop on pure and composite scaffolds surfaces is presented in Figure 5.
One minute after placing the drop on the surface of scaffolds, the contact angles of water drop with the surface of the scaffolds are shown in Table 1. According to the results, the water contact angel (WCA) decreased from 121 • for pure scaffold to 86 • for composite scaffold.

Bioactivity
To evaluate the bioactivity of the scaffolds, we immersed them in the SBF solution. After 4 weeks from immersion, the SEM images and EDX analysis represe-      The absorption of calcium for both pure and composite scaffolds decreased to less than 10 ppm after 2 weeks. However, after 4 weeks, the absorption of the composite scaffold was indiscernibly less than pure PHB, which means more bioactivity for the composite scaffold.

Cell attachment
According to the microscopic analysis, the cells were spherical and floated before culturing on cell culture dishes but after that, the cells were attached to the bottom of the dishes and they were spindle like. After 1 and 10 days of cell seeding on the scaffolds, the attachment and morphology of PDLSCs on the surface of pure and composite scaffolds was observed and evaluated by SEM ( Figure 10). SEM micrographs showed the cell attachment on both pure and composite scaffold. There was more attachment of PDLSCs on the surface of composite scaffold compared to pure scaffold, which can be clearly seen in SEM graphs.  In this study, we used MTT assay to assess and compare cell metabolic activities of both pure and composite scaffolds for 10 days. As can be seen in Figure 11, the viability of PDLSCs on the composite scaffold significantly increased compared to pure PHB.

Discussion
In the present study, we evaluated the in vitro and in vivo biocompatibility of pure PHB and PHB/1%CNTs composite scaffolds for PDL regeneration. First, we evaluated the structural and mechanical specifications in comparison with human PDL tissue. Presented SEM micrograph of electrospun fibers showed that the prepared fibers imitated the collagen fibrous structure in PDL, which consists of fibers with random orientation and connective pores that can provide a suitable space for the cells to proliferate and migrate. The mean fiber diameter in the composite scaffold increased, which is about twice compared to pure PHB fibers.  The definition of biomaterial is any material, which can be used as a part of live system for a specific time that aims to treat, replace, or regenerate any organ or tissue. To produce a scaffold for tissue engineering applications, the biomaterial must have some requirements, such as intrinsic biocompatibility, structural con-dition necessary for cell proliferation and migration, mechanical properties similar to the desired tissue [31].
However, most of the polymers are not eligible to provide the condition necessary for to create the scaffold to be used in tissue regeneration [32]. This increment might be caused by viscosity effect. This phenomenon is common and it confirmed that viscosity is an effective parameter during electrospinning [33]. The increment of the fiber diameter by addition the second material was reported in several studies [31]. The diameter of the scaffold pores is the only section of electrospinning method that is not easy to directly control. This can be indiscernibly controlled by obtaining smaller diameter fibers as smaller fibers result in smaller, more tightly packed pores [34][35]. However, it is not possible to alter the pore size without changing any of the other electrospinning parameters [33].
CNTs as the second material in the composite scaffold have led to improvement in the tensile strength.
The mechanical properties of the scaffold must be adapted to the specific tissue to guarantee the required mechanical functions during the formation of the new tissue [31]. Given that the target tissue is PDL, the tensile strength of human PDL was more than that obtained for pure PHB. By addition of only 1% of CNTs, the tensile strength has been almost equal to PDL, which is suitable for PDL regeneration. The noticeable increment of the mechanical specifications by small amounts of CNTs (mainly less than 2%) has been reported in numerous studies [36].
The wettability of the composite scaffold was also increased in the presence of CNTs. According to Yuan, et al. [37] a contact angle less than 90° indicates that wetting of the surface is favorable, and the fluid will spread over a large area on the surface, while the contact angles greater than 90° generally means that wetting of the surface is unfavorable, so the fluid will minimize its contact with the surface and form a compact liquid droplet [37].